BrdU-CLIP
CLIP with BrdU Affinity Purification
BrdU-CLIP sequences the binding sites of RBPs with single-nucleotide resolution. BrdU-CLIP fixes a major problem in HITS-CLIP where 5′ adapters are not attached to the cDNA due to premature termination of reverse transcription (Weyn-Vanhentenryck et al., 2014). BrdU-CLIP uses BrdUTPs and primers with 3′ and 5′ adapters during reverse transcription. This method enables greater cDNA yield and attaches both 5′ and 3′ adapters in a single step.
First, RNA-protein complexes are UV-crosslinked, digested with RNase, and immunoprecipitated. 3′ adapters are ligated to the RNA and reverse-crosslinked by proteinase K digestion. Some peptides from the crosslink remain bonded with the RNA strands, even after reverse-crosslinkage. RT is carried out using BrdUTPs and primers containing 3′ and 5′ adapters separated by an apurinic/apyrimidinic endonuclease (APE) cleavage site. The resultant cDNA is purified, circularized, and further purified by BrdU pull-down. Next, the cDNA is linearized by cleaving at the APE site, PCR-amplified, and sequenced.
Advantages:
- Sequences RNA-binding sites of RBPs with single-nucleotide resolution
- Captures both truncated and nontruncated cDNAs, unlike HITS-CLIP
- Provides higher cDNA yield due to BrdUTP
- Attaches 3′ and 5′ adapters in one RT step
Disadvantages:
- Tube-column transfers can result in major loss of material (Zarnegar et al., 2016)
- Not yet adopted widely by the scientific community
Reagents:
Illumina Library prep and Array Kit Selector
Reviews:
Scotti M. M. and Swanson M. S. RNA mis-splicing in disease. Nat Rev Genet. 2016;17:19-32
Vuong C. K., Black D. L. and Zheng S. The neurogenetics of alternative splicing. Nat Rev Neurosci. 2016;17:265-281
Hsiao Y.-H. E., Cass A. A., Bahn J. H., Lin X. and Xiao X. Global Approaches to Alternative Splicing and Its RegulationãRecent Advances and Open Questions. Transcriptomics and Gene Regulation. 2016;37-71
Bangru S. and Kalsotra A. Advances in analyzing RNA diversity in eukaryotic transcriptomes: peering through the Omics lens. F1000Research. 2016;5:2668
Flynn R. A., Martin L., Spitale R. C., Do B. T., Sagan S. M., et al. Dissecting noncoding and pathogen RNA-protein interactomes. RNA. 2015;21:135-143
References:
Weyn-Vanhentenryck S. M., Mele A., Yan Q., et al. HITS-CLIP and integrative modeling define the Rbfox splicing-regulatory network linked to brain development and autism. Cell Rep. 2014;6:1139-1152
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History: BrdU-CLIP
Revision by sbrumpton on 2017-06-21 09:06:28 - Show/Hide
CLIP with BrdU Affinity Purification
BrdU-CLIP sequences the binding sites of RBPs with single-nucleotide resolution. BrdU-CLIP fixes a major problem in HITS-CLIP where 5' adapters are not attached to the cDNA due to premature termination of reverse transcription (Weyn-Vanhentenryck et al., 2014). BrdU-CLIP uses BrdUTPs and primers with 3' and 5' adapters during reverse transcription. This method enables greater cDNA yield and attaches both 5' and 3' adapters in a single step.
First, RNA-protein complexes are UV-crosslinked, digested with RNase, and immunoprecipitated. 3' adapters are ligated to the RNA and reverse-crosslinked by proteinase K digestion. Some peptides from the crosslink remain bonded with the RNA strands, even after reverse-crosslinkage. RT is carried out using BrdUTPs and primers containing 3' and 5' adapters separated by an apurinic/apyrimidinic endonuclease (APE) cleavage site. The resultant cDNA is purified, circularized, and further purified by BrdU pull-down. Next, the cDNA is linearized by cleaving at the APE site, PCR-amplified, and sequenced.
Advantages:- Sequences RNA-binding sites of RBPs with single-nucleotide resolution
- Captures both truncated and nontruncated cDNAs, unlike HITS-CLIP
- Provides higher cDNA yield due to BrdUTP
- Attaches 3' and 5' adapters in one RT step
Disadvantages:- Tube-column transfers can result in major loss of material (Zarnegar et al., 2016)
- Not yet adopted widely by the scientific community
Reagents:Illumina Library prep and Array Kit SelectorReviews:Scotti M. M. and Swanson M. S. RNA mis-splicing in disease. Nat Rev Genet. 2016;17:19-32Vuong C. K., Black D. L. and Zheng S. The neurogenetics of alternative splicing. Nat Rev Neurosci. 2016;17:265-281Hsiao Y.-H. E., Cass A. A., Bahn J. H., Lin X. and Xiao X. Global Approaches to Alternative Splicing and Its RegulationãRecent Advances and Open Questions. Transcriptomics and Gene Regulation. 2016;37-71Bangru S. and Kalsotra A. Advances in analyzing RNA diversity in eukaryotic transcriptomes: peering through the Omics lens. F1000Research. 2016;5:2668Flynn R. A., Martin L., Spitale R. C., Do B. T., Sagan S. M., et al. Dissecting noncoding and pathogen RNA-protein interactomes. RNA. 2015;21:135-143References:Weyn-Vanhentenryck S. M., Mele A., Yan Q., et al. HITS-CLIP and integrative modeling define the Rbfox splicing-regulatory network linked to brain development and autism. Cell Rep. 2014;6:1139-1152